Improve Embryo Quality Research Articles

Clinical validation of the early embryo viability assessment system: Analysis for the blastocyst morphology and pregnancy outcomes

Objective: To determine the relationship between the early embryo viability assessment (EEVA) and blastocyst morphological parameters and pregnancy outcomes. Methods: This retrospective cohort study was conducted on 291 intracytoplasmic sperm injection cycles including 2 522 embryos with indications of prolonging embryo culture to the blastocyst stage in the Genea embryo review incubator, and 511 single vitrified- warmed blastocyst transfer cycles from January 2020 to June 2023. The EEVA system produced an EEVA score from E1 (best) to E5 (worse) for the potential of blastocyst formation. Blastocyst morphology was evaluated. The association between the EEVA score and each type of blastocyst morphology, implantation rate, clinical pregnancy, and ongoing pregnancy were assessed using generalized estimating equations. Results: The inner cell mass A (ICM A), trophectoderm A (TE A), blastocoele expansion degree of 3, 4, 5, 6, 7 rates were higher with lower the EEVA score. The adjusted odd ratio (aOR) (E5 vs E1) was 0.3 for ICM A, 0.174 for TE A and 0.210 for BL3, 4, 5, 6, 7 (all P<0.001), suggesting a significant association between lower EEVA scores and improved embryo quality. The implantation, clinical pregnancy, and ongoing pregnancy rate were also higher with lower the EEVA score. The aOR of E5 vs E1 was 0.245 for implantation, 0.185 for clinical pregnancy and 0.200 for ongoing pregnancy rate (P<0.001). Conclusions: There were associations between blastocyst morphology, pregnancy outcome and EEVA scores. The good blastocyst morphology and pregnancy outcomes are higher with lower the EEVA score.

Reproductive outcomes in fresh transfer cycles and antagonists with premature luteinizing and/or progesterone surge: a single center retrospective cohort study.

The optimal outcome of assisted reproductive technology is a successful live birth after fresh embryo transfer. However, the success pregnancy rate of fresh embryo transfer cycle in antagonist protocol is lower than that observed in other protocols. Despite the use of antagonists (GnRH-ant), the incidence of luteinizing hormone surge and elevated progesterone levels remain at approximately 5%-38%. Progesterone is widely recognized to exert adverse effects on fresh embryo transfer outcomes. This study aimed to investigate the impact of luteinizing hormone surge and progesterone levels on live birth rate following fresh embryo transfer and explore appropriate progesterone thresholds to enhance pregnancy outcomes. This retrospective cohort study included a total of 1,177 antagonist protocol cycles with fresh embryo transfer. The patients were divided into four groups based on the presence of premature LH surge and progesterone level on trigger day>1.5ng/ml. Then, the relationship between the variables and the pregnancy outcome was analyzed and compared in each group. The transient rise of luteinizing hormone did not impact pregnancy outcomes (P=0.345; P=0.3; P=0.787), in contrast to progesterone levels on the day of hCG administration (P=0.047*; P=0.015*; P=0.021*). In cases with luteinizing hormone surge, elevated progesterone levels were correlated with higher antral follicle count (AFC), and as progesterone levels increased, a greater quantity of oocytes and embryos were obtained. However, there was no statistically significant difference in pregnancy outcomes. In cases without luteinizing hormone surge, elevated progesterone levels led to significantly poorer pregnancy outcomes. Furthermore, the curve-fitting and threshold-effect analysis revealed a notable decline in live birth rates when progesterone exceeded or equaled 1.10ng/ml (OR, 0.25; 95% CI, 0.09-0.66; P = 0.005*). The GnRH-ant dosage addition should be carefully selected in flexible antagonist protocols. The presence of elevated progesterone levels may be associated with improved embryo quality when luteinizing hormone surge occurred. In the absence of a luteinizing hormone surge, progesterone levels showed a larger impact on the pregnancy outcome, and fresh embryo transfer should not be performed if the progesterone level on the day of hCG administration is higher than 1.10ng/ml.

Temperature Dynamics in Early Pregnancy: Implications for Improving In Vitro Fertilization Outcomes

In assisted reproductive technology, in vitro fertilization involves cultivating embryos in an artificial environment, often yielding lower-quality embryos compared to in vivo conditions. This study investigated core body temperature (CBT) fluctuations in mice during early pregnancy. Their CBT was measured with a high temporal resolution to identify the optimal thermal conditions during the first five days post-fertilization, aiming to improve in vitro culture conditions. Data were collected from 12 female mice, with 8 becoming pregnant, using temperature loggers every minute for 11 days. Data analysis focused on trends, circadian rhythms, frequency components, and complexity using multiscale entropy (MSE). The results for the pregnant mice showed a mean CBT increase from 37.23 °C to 37.56 °C post-mating, primarily during the light phase, with a significant average rise of 0.58 °C. A Fourier analysis identified dominant 24, 12, 8, and 6 h components, with the 24 h component decreasing by 57%. Irregular fluctuations decreased, and MSE indicated increased complexity in the CBT time series post-mating. These results suggest that reducing diurnal temperature variations and maintaining a slightly elevated mean CBT of approximately 37.5 °C, with controlled minor fluctuations, may enhance embryo quality in pregnant mice. This study provides a reference for temperature regulation in embryo culture, improving embryo quality by aligning in vitro conditions with the natural thermal environment of the fallopian tubes.

P-111 Artificial Oocyte Activation (AOA) through piezoelectric stimulation post-Intracytoplasmic Sperm Injection (ICSI) for teratozoospermia patients

Abstract Study question Does employing piezoelectric stimulation on oocytes post-ICSI in teratozoospermia cases affect early embryonic development rates? Summary answer In a retrospective study, piezoelectric stimulation after ICSI showed increased fertilization rates and improved embryo quality in teratozoospermia cases. What is known already Fertilization can fail due to various factors. One of the causes of fertilization failure after ICSI is the inability to achieve oocyte activation, a process initiated by sperm-induced events. Piezoelectric stimulation, a method of artificial oocyte activation, involves applying specific electric currents post-ICSI, elevating Ca+2 levels. Studies suggest its efficacy in enhancing fertilization rates and improving embryo quality, particularly in cases of total fertilization failure and severe sperm abnormalities. However, available literature on the efficiency of electrical stimulation in cases of sperm abnormalities is limited. Study design, size, duration In this retrospective study conducted at our reproductive center between January 2023 and October 2023, we enrolled 122 patients diagnosed with male factor infertility. These patients were divided into two groups: the control group (n = 61) where piezoelectric stimulation was not employed post-ICSI, and the AOA group (n = 61) where piezoelectric stimulation was utilized after the ICSI procedure. Participants/materials, setting, methods The study assessed 2122 oocytes, with 1031 in the control and 1091 in the AOA group. Piezoelectric stimulation was administered oocytes approximately 20-30 minutes after ICSI. The quality of sperm samples was assessed based on Kruger’s strict criteria. Following ICSI, statistical analysis utilized Student’s t-test and chi-squared tests to assess the impact of piezoelectric stimulation on fertilization and embryo quality, considering p < 0.05 as significant and p < 0.001 as highly significant. Main results and the role of chance Clinical outcomes were compared between two groups. No significant differences were observed between the control group and AOA group regarding female age (29.93±4.9 vs. 29.93±5.11, p = 0.988) and male age (34.25±3.45 vs. 33.93±3.64, p = 0.628), the number of retrieved oocytes (16.90±11.04 vs. 17.89±9.3, p = 0.344), the number of metaphase II (11.02±6.8 vs.12.46±6.6, p = 0.162). However, a highly significant difference was noted in the distribution of fertilization rate (59.08% [397/672] vs. 66.84% [508/760], p = 0.002), high-quality embryos rate (day 3) (44.08% [175/397] vs. 54.33% [276/508], p = 0.002), blastocyst rate (40.05% [159/397] vs. 50% [254/508], p = 0.003) and the good blastocyst development rate (14.11% [56/397] vs. 24.41% [124/508], p = 0.0001). Cleavage rate was significantly higher in the AOA group (85.24% [433/508]) than control group (79.09% [314/397]), p = 0.01. Limitations, reasons for caution Constraints include the study’s retrospective, single-center design, and a limited sample size. Wider implications of the findings Piezoelectric stimulation demonstrates effectiveness in improving fertilization rates and embryo quality in teratozoospermia cases after ICSI. Trial registration number not applicable

P-178 Putrescine supplementation enhances embryonic development in patients with Recurrent Implantation Failure (RIF)

Abstract Study question Does putrescine, a type of polyamine, affect embryonic development in patients with recurrent implantation failure (RIF)? Summary answer Putrescine supplementation on cumulus-oocyte complexes (COCs) was associated with better embryologic development from fertilization to blastulation in patients with RIF. What is known already Putrescine, a type of polyamine, plays a role in cell growth and proliferation. Several animal studies suggest the potential impacts of putrescine on embryo development, but its effects on human IVF have yet to be extensively studied. Study design, size, duration This retrospective study analyzed 442 patients who have undergone IVF cycles three or more times from January to December 2023. The average IVF cycle number of RIF patients was 7.4 times. A total of 1356 oocytes from the putrescine group and 1391 oocytes from the control group underwent aspiration and ICSI. Further analysis was conducted by classifying RIF patients into two groups: patients under 40 years of age and patients over 40 years of age. Participants/materials, setting, methods COCs were cultured for 4 hours in a culture medium supplemented with 0.5mM putrescine. 2 hours after oocyte collection, cumulus cells were denuded, and ICSI was performed. Embryonic development was evaluated based on fertilized oocytes (2PN), good quality embryos (GQE) on day 3, and blastulation rates on day 5/6. Group differences were analyzed using t-tests and chi-square tests (p < 0.05). Main results and the role of chance When maternal age was ≥40, the fertilization rate in the putrescine group was significantly higher than that in the control group (86.6% vs. 80.5%, p=0.002). The putrescine group had a numerically higher proportion of embryos with top-grade morphology on day 3 (47.8%, compared with 53.3% for control; p=0.057), without statistical significance. Additionally, the rate of blastulation on day 5 indicated a significant difference between the two groups (14.5% vs. 6.1%, p=0.0001). Further analysis was conducted in younger patients (<40 years old), and it also showed a significantly higher fertilization rate (89.7% vs. 71.1 p=0.0001) and blastocyst development rates on the day 5 in the putrescine group (22.3% vs. 11.1%, p=0.0001), respectively. This study suggests that putrescine supplementation can be utilized to improve embryo quality and facilitate blastocyst formation in IVF procedures for patients with RIF, irrespective of age. Limitations, reasons for caution Putrescine supplementation in IVF showed improved fertilization and blastulation rates, but caution is needed due to the small sample size used. Therefore, a larger sample size will help improve the robustness of the conclusions drawn with respect to the embryologic development in patients with RIF. Wider implications of the findings The study’s findings suggest that putrescine supplementation in IVF may enhance outcomes for patients with RIF, potentially leading to personalized treatments. Trial registration number not applicable

P-196 Early blastocyst formation on day 4 is associated with improved clinical outcome and advocates for embryo quailty assessment on day 4

Abstract Study question Is early cavitation on day 4 associated with improved clinical outcome? Summary answer Early blastulation on day 4 is a positive predictor of implantation after single blastocyst transfer on day 5. What is known already In order to select the blastocyst with the highest implantation potential for transfer or freezing, several morphological and/or morphokinetic parameters have been proposed. For instance, early compaction on day 3 (D3) or blastocyst expansion on day 5 (D5) have been reported to be relevant when selecting embryos for transfer. However, day 4 has often been overlooked and very few studies focused on early blastulation on day 4 (D4). Indeed, data on early blastulation on day 4 is scarce. It was however recently demonstrated that embryos transferred on day 4 could provide acceptable pregnancy rates. Study design, size, duration This monocentric retrospective cohort study was conducted in all consecutive couples undergoing IVF cycles in 2017-2022 with single blastocyst transfer and observation on day 4. Participants/materials, setting, methods Morphological and morphokinetic assessment was performed by 2 operators at 93±2h, 116±2h (and 140±2h) post insemination, on day 4, 5 (and 6) respectively. A total of 26 507 D4 embryos were included in this study and grouped according to the observation of early cavitation or not : 23 244 were in the D4 control group (absence of early cavitation on D4) and 3263 were in the D4 blastocyst group (observation of early cavitation on D4). Main results and the role of chance The proportion of embryos used clinically for either transfer or freezing on day 5 or 6 was significantly higher in the D4 blastocyst group than in D4 control group (86.7% vs 50.1% respectively, p < 0.05). Concerning pregnancy rate, cinical pregnancy rate was significantly higher in the D4 blastocyst group than in D4 control group (p < 0.05). In the D4 control group, 5051 embryos were transferred and clinical pregnancy rate is 28.7%. In the D4 blastocyst group, 1286 embryos were transferred (fresh or frozen) and clinical pregnancy rate is 40.2%. Limitations, reasons for caution The retrospective and monocentric design calls for caution when considering the generalizability of our findings. Wider implications of the findings We postulate that D4 embryo quality assessment should be widely implemented in any blastocyst program in order to improve embryo quality assessment and subsequently clinical outcome. Trial registration number not applicable

P-639 Are GnRH-agonists (GnRH-a) safer and more effective ovulation triggers than human chorionic gonadotrophin (hCG)? A retrospective analysis of frozen embryo replacement cycle (FERC) outcomes

Abstract Study question Are GnRH-agonists (GnRH-a) safer and more effective ovulation triggers than human chorionic gonadotrophin (hCG)? A retrospective analysis of frozen embryo replacement cycle (FERC) outcomes Summary answer GnRH-a trigger reduces the incidence and severity of ovarian hyperstimulation syndrome (OHSS) leading to significantly improved clinical outcomes when compared to hCG trigger for FERCs. What is known already Ovulation induction plays a crucial role in the success and safety of ART cycles. hCG is extensively used as an alternative to LH to trigger ovulation. However, high levels of sustained hCG exposure increases the risk of OHSS, a potentially life-threatening complication of ART. To eliminate the risk and incidence of OHSS, GnRH-a can be used to trigger ovulation. Although there are concerns regarding clinical outcomes with fresh transfers following GnRH-a trigger, it elicits a more physiological response compared to hCG. This may lead to improved embryo quality, utilisation, and better clinical outcomes following a freeze-all strategy, reducing patients’ time-to-pregnancy. Study design, size, duration This was a retrospective data analysis of FERC outcomes following fresh IVF or ICSI cycles, involving ovulation induction using either hCG or GnRH-a as a final trigger. The evaluation included results from cycles spanning an eight-year period, from January 2012 to December 2019 at Hammersmith Hospital London. A total of 160 cycles were included in the study, with 76 in the hCG trigger group and 84 in the GnRH-a trigger group. Participants/materials, setting, methods Strict inclusion criteria were employed to render the study groups as comparable as possible and to minimise the effect of independent variables, such as oocyte and sperm factors, on oocyte or embryo developmental competence. Inclusion criteria: women ≤37 years old at the start of treatment, GnRH antagonist stimulation, blastocyst vitrification-warming and transfers only. Statistical significance was calculated using independent t-test and Fisher’s Chi-square exact test. Main results and the role of chance The mean age (±SD) of patients at the time of oocyte retrieval was comparable across groups. The average number of oocytes collected were significantly higher in the GnRH-a group compared to hCG (28.31 vs. 18.07, respectively, P < 0.001). Embryological data (maturation, fertilisation, blastocyst formation, blastocyst utilisation and good quality blastocyst formation rates) were comparable across both groups. OHSS rate was significantly lower in the GnRH-a group (17% vs. 50%. P < 0.0001). The following clinical data were significantly higher (P < 0.05) in the GnRH-a group compared to hCG: implantation rate (56% vs. 36%), clinical pregnancy rate (56% vs. 36%) and live birth rate (42% vs. 27%). Limitations, reasons for caution The biggest limitation of this study is its retrospective nature and the application of various inclusion-exclusion criteria that have resulted in relatively small sample sizes. Additionally, numerous other confounders, e.g. patient body mass index, cause/duration of infertility and stimulation duration, have not been considered in this study. Wider implications of the findings The findings of this study are promising and demonstrate a great potential for benefit in the use of GnRH-a trigger with a freeze-all strategy to minimise the risk of OHSS, diminishing the associated patient morbidity and mortality, reducing healthcare costs, improving clinical outcomes, and reducing the overall time-to-pregnancy for patients. Trial registration number not applicable

P-123 Comparison of the performance of 3 models (iDAScore v1.0, KIDScore D5 v3, and NEQsi score) for predicting pregnancy after single blastocyst transfer

Abstract Study question What is the respective performance of an AI model, a morphokinetic model and an embryo morphology model for the prediction pregnancy after single blastocyst transfer? Summary answer KIDScore™ D5 v3, NEQsi score, and iDAScore™ v1.0 demonstrated similar performance in predicting biochemical pregnancy, clinical pregnancy, and live birth. What is known already Blastocyst selection for transfer currently mostly relies on Gardner’s morphological classification, which suffers from relatively limited predictive performance and interoperator variability. Both morphokinetic scores and the development of AI tools raised hope for improved embryo quality assessment in IVF. The NEQsi score is a very recently published digital transcription of the Gardner classification, allowing easier data categorization and comparison with other scores / models. The respective performance of these models / scores has hardly been reported up to now. Studying this in a setting with systematic single blastocyst policy allows avoiding biases related to patients’ selection. Study design, size, duration This is a retrospective monocentric study conducted in 2020-2022 on single blastocyst transfers after culture in a time-lapse incubator (Embryoscope©). All embryos with a known clinical outcome after fresh or frozen transfer and with KIDScore™ D5 v3, NEQsi score, and iDAScore™ v1.0 were included, regardless of the ART technique. Participants/materials, setting, methods All patients gave their consent for anonymous analysis of their clinical data. All scores were compared by conducting a One-way ANOVA test followed by a non-parametric Kruskal-Wallis test. A ROC curve was calculated for the prediction of both biochemical, clinical pregnancy and live birth. Main results and the role of chance 1888 single blastocyst transfers were performed in 1223 patients, 907 resulting in a biochemical pregnancy, 626 in a clinical pregnancy, and 495 in a live birth. The mean values were 7.00 (± 1.64) for the KIDscore, 7.99 (± 1.30) for the NEQsi, and 8.50 (± 1.00) for the iDAscore. The mean score values were significantly different between groups according to positive or negative clinical outcome (p < 0.0001 for all). The ROC curves showed that all 3 scores are predictive of a biochemical pregnancy (AUC KIDScore = 0.62; AUC NEQsi = 0.63; AUC iDAScore = 0.60), clinical pregnancy (AUC KIDScore = 0.64; AUC NEQsi = 0.65; AUC iDAScore = 0.61), and a live birth (AUC KIDScore = 0.62; AUC NEQsi = 0.63; AUC iDAScore = 0.60). These AUCs were not significantly different, and none of these scores appeared to be superior to the others in predicting live birth after single blastocyst transfer. Limitations, reasons for caution The retrospective and monocentric design calls for caution when generalizing these results. Wider implications of the findings The respective relevance of these scores in shortening time to pregnancy by ranking usable embryos within a cohort should be considered in further studies. Trial registration number not applicable

Optimizing IVF Success: A Case Study of Melatonin-Assisted Frozen Embryo Transfer for Poor Oocyte Quality and Endometriosis.

The case study investigates the journey of a couple facing infertility. It intensifies the challenges, including poor oocyte quality and endometriosis. In spite of two failed in vitro fertilization cycles, the decision for ovum pickup (OPU) was made, followed by intra-cytoplasmic sperm injection (ICSI), embryo treatment with melatonin, and frozen embryo transfer (FET) to optimize the chances of a successful pregnancy. The couple opted for this approach. OPU yielded four poor-quality oocytes, prompting ICSI and melatonin treatment to enhance embryo quality. The embryos were exposed to culture supplementation with melatonin for 72 hours before being transferred to conventional media. After 5 days or 120 hours, the embryos developed into 3BB quality blastocysts, indicative of developmental stage and morphology. The blastocysts were then cryopreserved, and after 2 months, FET was conducted, resulting in the transfer of two embryos, which subsequently led to a positive pregnancy indication, as indicated by a β-hCG level of 233 mUI/ml measured 14 days post transfer. This approach highlights the effectiveness of melatonin supplementation in improving embryo quality and ultimately facilitating successful pregnancy in complex scenarios like endometriosis-related infertility.

MCT1-mediated transport of valeric acid promotes porcine preimplantation embryo development by improving mitochondrial function and inhibiting the autophagic AMPK-ULK1 pathway

Oocytes and embryos are highly sensitive to environmental stress in vivo and in vitro. During in vitro culture, many stressful conditions can affect embryo quality and viability, leading to adverse clinical outcomes such as abortion and congenital abnormalities. In this study, we found that valeric acid (VA) increased the mitochondrial membrane potential and ATP content, decreased the level of reactive oxygen species that the mitochondria generate, and thus improved mitochondrial function during early embryonic development in pigs. VA decreased expression of the autophagy-related factors LC3B and BECLIN1. Interestingly, VA inhibited expression of autophagy-associated phosphorylation-adenosine monophosphate-activated protein kinase (p-AMPK), phosphorylation-UNC-51-like autophagy-activated kinase 1 (p-ULK1, Ser555), and ATG13, which reduced apoptosis. Short-chain fatty acids (SCFAs) can signal through G-protein-coupled receptors on the cell membrane or enter the cell directly through transporters. We further show that the monocarboxylate transporter 1 (MCT1) was necessary for the effects of VA on embryo quality, which provides a new molecular perspective of the pathway by which SCFAs affect embryos. Importantly, VA significantly inhibited the AMPK-ULK1 autophagic signaling pathway through MCT1, decreased apoptosis, increased expression of embryonic pluripotency genes, and improved embryo quality.

Effect of coenzyme Q10 supplementation on post-vitrification mouse embryo development

Objective: To investigate the effects of coenzyme Q10 (CoQ10) supplementation on post-vitrification embryo development and gross morphology. Methods: Balb/c mouse embryos were cultured in potassium simplex optimised medium (KSOM) with varying CoQ10 concentrations [0 (control), 20, 40, and 60 μΜ]. The most effective CoQ10 concentration (40 μM) was selected for subsequent post-vitrification morphology study. Embryos were randomly divided into four groups: Group A (non-vitrified without CoQ10), Group B (non-vitrified with CoQ10), Group C (vitrified without CoQ10), and Group D (vitrified with CoQ10), followed by vitrification at the 8-cell stage. Survival rates and development until the blastocyst stage were evaluated through morphological examinations using ASEBIR's system, distinguishing normal and abnormal embryos. Results: Supplementation of 40 μM CoQ10 significantly increased blastocyst formation (95%) compared to the control group (92%), 20 μM (62%), and 60 μM (56%) (P<0.001). Following vitrification, Group D exhibited a significant increase in blastocyst formation (92%) compared to Group C (82%) (P<0.05). Morphological assessments indicated superior embryo quality in Group B over Group D during the cleavage stage, morula, and blastocyst (P<0.05). Conclusions: CoQ10 supplementation exhibits promising potential to enhance preimplantation embryo development, increase blastocyst formation rates, and improve embryo quality post-vitrification. This offers a promising approach to mitigate oxidative stress on embryos, potentially improving overall assisted reproductive technology outcomes.

MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway

BackgroundExtracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-β pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality.MethodsZygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-β pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses.ResultsWe demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it’s a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups.ConclusionsOur findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-β signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

Oocyte activation with phospholipase Cζ mRNA induces repetitive intracellular Ca2+ rises and improves the quality of pig embryos after intracytoplasmic sperm injection.

For the intracytoplasmic sperm injection (ICSI) procedure in pigs, an electrical pulse (EP) has been used as an effective method for oocyte stimulation, but unlike sperm, EP is unable to induce Ca2+ oscillations. In this study, we investigated the effects of generating artificial Ca2+ oscillations with phospholipase Cζ (PLCζ) mRNA, a candidate sperm factor, on fertilization, embryonic development, and gene expression after ICSI. Firstly, the concentration of PLCζ mRNA of a fixed volume (1.0 pl) that would induce a pattern of Ca2+ rise similar to that of in vitro fertilized (IVF) sperm was examined and determined to be 300 ng/μl. Secondly, the effects of oocyte stimulation methods on fertilization and embryonic development were investigated. ICSI-oocytes were activated by EP (EP group) or by PLCζ mRNA (PLCζ group). Furthermore, IVF-oocytes (IVF group) and ICSI-oocytes with and without an injection of buffer (buffer and untreated groups, respectively) were used as controls. It was found that the rates of normal fertilization in the PLCζ and EP groups were significantly higher than those in the buffer and untreated groups. The blastocyst formation rates did not differ among the groups. The embryo quality in the EP group was inferior to those in the PLCζ and IVF groups. Additionally, the expression level of a proapoptosis-related gene (Caspase-3) in the EP group was significantly higher than those in the PLCζ and IVF groups. Our data suggest that oocyte activation by PLCζ mRNA has the effect of improving embryo quality.

Excluding alanine from minimum essential medium (MEM) nonessential amino acid supplementation of the culture medium facilitates post-fertilization events and early cleavages of bovine oocytes fertilized in vitro.

Embryonic transfer of bovine blastocysts produced by in vitro fertilization is widely utilized-despite a compromised conception rate. It has been suggested that a set of four evaluation criteria for judging the quality of embryos, based on the timing of early cleavages and proper morphologies of embryos, can effectively predict pregnancy success. These blastocysts are hereafter referred to as four-criteria-compliant blastocysts. The same criteria should be used to modify the culture media to improve embryo quality. For example, culture media is often supplemented with nonessential amino acids (NEAA) at a uniform concentration despite the major variation in their concentration in the oviductal fluid. In the present study, the effects of the embryo culture medium, namely CR1, supplemented with all seven MEM NEAA or six of them, excluding one at a time, were examined. All media, except for the medium that did not contain proline and serine, tended to improve the efficiency of producing four-criteria-compliant blastocysts, and excluding alanine was particularly effective. The absence of alanine resulted in the rapid occurrence of the first cleavage and pronuclear formation of fertilized oocytes in the alanine-free medium compared to that in the medium containing alanine. These results suggested that alanine hinders certain events involved in the progression of early embryogenesis, which is necessary to achieve the four criteria that provide a benchmark for pregnancy. Therefore, a significantly higher percentage of embryos satisfied the recommended criteria and developed into four-criteria-compliant blastocysts when developed in alanine-free medium than in alanine-containing medium.

Modification of cryoprotectants on sperm cryopreservation: A study of embryo development.

This research was conducted to analyze the effect of cryopreservation of sperm using modified cryoprotectants on embryo development through in vitro fertilization (IVF). In this research, three types of cryoprotectant combinations were compared, including Nakagata, modified cryoprotectant, and commercial (Kitazato). Several parameters, namely sperm concentration, motility, morphology, plasma membrane integrity, cryo-survival rate, and viability rate, were measured and compared before vitrification and warming. Embryo development was also observed on the first and third days of development based on the cell number, cell size, and fragmentation rate. Sperm cryopreservation exhibited a negative influence on embryo quality. Both Nakagata cryoprotectants and modified cryoprotectants attained good-quality embryos. In terms of embryonic development, this research revealed a modified cryoprotectant superior to Nakagata's cryoprotectant, although Kitazato was superior to the Nakagata cryoprotectant and modified cryoprotectant. Significant differences were found in the three cryoprotectants on observations on day 1 and day 3, all of them with p-value < 0.05. Modified cryoprotectant was found to be better than Nakagata but less significant than Kitazato in terms of embryonic development quality. Therefore, modified cryoprotectants could be a better alternative compared to commercial (Kitazato) cryoprotectants for improving embryo quality.

Effect of additional cytoplasm injection on the cloned bovine embryo organelle distribution and stress mitigation

Although somatic cell nuclear transfer (SCNT) is a critical component of animal cloning, this approach has several issues. We previously introduced the cytoplasm injection cloning technology (CICT), which significantly improves the quality and quantity of cloned embryos. This study examined the residual status of fused cytoplasmic organelles, such as the endoplasmic reticulum (ER) and lysosomes, in the CICT group during early embryo development. We found that extra-cytoplasmic organelles stained using the ER-Tracker™ Green dye and LysoTracker™ Deep Red probe were fused and dispersed throughout the recipient oocyte and were still visible in day 8 blastocysts. We screened for ER stress, autophagy, and apoptosis-related genes to elucidate the association between the added organelles and improved embryo quality in CICT-cloned embryos. We found that CHOP, ATF4, ATG5, ATG7, and LC3 genes showed non-significantly up- or downregulated expression between CICT- and in vitro fertilization (IVF)-derived embryos but showed significantly (p < 0.05) upregulated expression in SCNT-cloned embryos. Surprisingly, a non-significant difference in the expression of some genes, such as ATF6 and caspase-3, was observed between the CICT- and SCNT-cloned embryos. Our findings imply that compared to conventional SCNT cloning, CICT-derived cloned embryos with additional cytoplasm have much higher organelle activity, lower autophagy, lower rates of apoptosis, and higher embryo development rates.

Single Step, Two-Steps, and Microfluidics in Embryo Culture. How Do They Measure up in Improving Embryo Quality?

The issues surrounding the culture of the preimplantation human embryo are distinct from those associated with the propagation of somatic cells, given that the starting point of culture, the pronucleate oocyte, and the final stage of development, the blastocyst, are as distinct as any two cell types can be with regards to their nutrient requirements and metabolism. Furthermore, the cleavage stage period of embryo development is exquisitely sensitive to its environment and has limited capacity to regulate against disruptions in its surroundings, especially changes in pH. Historically the media used in human IVF, such as Human Tubal Fluid medium, were based on simple salt solutions but lacked key regulators of blastomere function, specifically amino acids. A resurgence of research in embryo metabolism and physiology in the 1980s paved the way for the development of Sequential media, designed not only to accommodate the changing requirements of the embryo itself (both in terms of carbohydrates and amino acids), but which were also based on available data on the dynamic/differing composition of the Fallopian tube and uterus. Sequential media, especially when used in combination with isolettes, were shown to be highly effective in supporting blastocyst development and significantly increased implantation rates, thereby facilitating the worldwide movement to single embryo transfer. However, while this approach partially satisfied the changing nutrient requirements of the embryo, and further facilitated the removal of toxins built up in the culture drop over 48h, such as ammonium, it necessitated moving embryos. Unless undertaken using warmed gassed isolettes, moving embryos inevitably resulted in shifts in temperature and pH. With the advent of time-lapse, there began a growing movement towards the use of one-step media in order to keep the embryo within an imaging incubator. To facilitate this, hybrid type media were formulated, and while they do not accommodate the dynamic requirements of the preimplantation period, their use meant embryos were not exposed to changes in either temperature or pH. To date, clinical data have reported similar outcomes using both media approaches, plausibly due to a trade-off of the inherent limitations of either approach. An alternative approach proposed decades ago, is to provide the embryo with a gradient of nutrients within a perfusion system, thereby facilitating a dynamic culture environment without the need to move embryos. Unfortunately, until recently, the technology to support this concept has not been available. However, the recent development of 3D printing using 2-photo lithography, has made it possible to create embryo-specific perfusion systems. Hence, we are now able to evaluate the potential of such dynamic systems and could find ourselves on the verge of a new era in embryo culture.

#154 : The Creation of a New Embryo Culture Medium with 31 Optimal Components Derived from Human Oviduct Fluid: A Prospective Multicenter Randomised Trial

Background and Aims: Although culture conditions were recognized as key points, an optimal culture medium formulation, which closely resembles natural human oviduct fluid (HOF), has not been obtained yet. Our aim is to make an ideal embryo culture medium close to HOF components, and to evaluate the quality of this medium with embryo quality and clinical outcomes in assisted reproductive technology (ART) by a prospective randomized controlled trial (RCT). Method: Study I: HOF was collected laparoscopically from patients (n = 28) with normal pelvic findings. According to HOF analysis results, the new medium ‘OVIT’ was designed. Study II: Embryos (2 pronuclei (2PN) = 9,633) were assigned from 1,435 patients. The blastulation rate (BR), good BR (gBR), utilized (transferred/cryo-preserved) BR (uBR), pregnancy rate (PR), and miscarriage rate (MR) were compared between the OVIT and control groups by RCT. Study III: The neonatal healthiness who were concepted by ART using OVIT and /or control medium was compared between two groups. Results: The novel medium ‘OVIT’ was produced according to 31 HOF components. The concentrations of essential amino acids (e-AAs) were lower in OVIT than in current media, yet the opposite was true for non-e-AA concentrations. gBR and uBR were higher in the OVIT group than in the control group. In the older female group, gBT and uBR were significantly higher in the OVIT group. The neonatal conditions of those who were born by ART using OVIT and/or control medium were similar. Conclusion: HOF components and AA concentrations were analysed, and the results indicated numerous differences between OVIT and current media. This medium provides a better culture condition and improves embryo quality. This study has found novel perspectives for older patients having difficulties in producing embryos of good quality and indicated the superior quality of OVIT compared with the quality of traditional media.

#289 : Mitochondrial Activator BGP-15 Protects Sperm from DNA Damage During Sperm Recovery for Assisted Reproductive Technology

Background and Aims: The recovery of sperm by assisted reproductive technology (ART) results in significant DNA damage. Elevated levels of DNA damage in sperm have been linked to poor-quality embryos and increased pregnancy loss after ART. Therefore, in this study, we aimed to investigate potential of the new mitochondrial activator BGP-15 to prevent sperm DNA damage in an in vitroejaculate over time. Additionally, we sought to determine whether BGP-15 can preserve sperm quality during various sperm recovery techniques used in ART. Method: We incubated donated human semen specimens untreated or treated with BGP-15, then examined sperm motility and DNA damage (DNA fragmentation and oxidation). Semen samples were then processed using different clinical sperm recovery methods: simple wash (SW), swim-up (SU) and density gradient centrifugation (DGC); and the purified sperm underwent further analysis including motility, vitality, ROS levels, mitochondrial membrane integrity and mitochondrial membrane potential (MMP), and DNA damage. Results: Semen samples incubated with BGP-15 demonstrated improved sperm motility and reduced DNA damage levels. SW samples had the highest sperm count, while SU had the highest vitality and DNA integrity, but the lowest MMP. DGC sperm had increased MMP after BGP-15 Treatment. Although BGP-15 treatment only reduced sperm DNA fragmentation in SW samples, it reduced DNA oxidation in all recovery methods. Conclusion: Our findings indicate that clinical manipulation of patient semen samples can negatively impact sperm quality, and different sperm recovery methods have unique effects. However, the addition of BGP-15 to clinical processes can preserve sperm quality, potentially leading to improved embryo quality and ART success.

#149 : Improved Embryo Quality of Infertile Women at Different Ages by Oral Administration of an Antioxidant Mixture

Background and Aims: Oxidative stress is known to affect oocyte/embryo quality. In the previous meeting of ASPIRE, an antioxidant mixture (AOM) developed by us was shown to improve oocyte/embryo quality. Since oxidative stress is related to aging of body, AOM may be able to support the oocyte/embryo quality of women at advanced ages. We therefore examined the effects of AOM administration on the oocyte/embryo quality of women at different ages. Method: AOM supplement was given orally for at least one and half month (at most 7 months) to infertile women who had participated in at least one IVF/ICSI program and whose quality of embryos was poor or fair. After the supplementation period, oocyte-pickup was carried out, and either IVF or ICSI was subjected to the patients. Then, the data of oocyte maturity, fertilization, developmental capacity, and bioavailability of embryos (No. of embryos transferred and cryopreserved) were collected retrospectively. Results: The effects of AOM on the embryo quality of women at different ages were compared (Age groups were: [Formula: see text] 30yr; 31 [Formula: see text] 35yr; 36 [Formula: see text] 40yr; 41yr[Formula: see text]). In all groups, oocyte retrieval and maturation rates were slightly increased. More evidently, the developmental capacity, especially blastocyst formation, was significantly improved by AOM in all groups ([Formula: see text] 30yr: 20.0% vs 63.6%; 31 [Formula: see text] 35yr; 30.8% vs 68.9%; 36 [Formula: see text] 40yr: 36.4% vs 60.8%; 41yr[Formula: see text]: 36.4% vs 52.9%). Accordingly, the number of embryos cryopreserved was markedly increased ([Formula: see text] 30yr: 0.3±0.6 vs 1.8±2.4; 31 [Formula: see text] 35yr; 0.7±0.9 vs 1.4±1.2; 36 [Formula: see text] 40yr: 0.6±0.8 vs 1.1±1.0; 41yr[Formula: see text]: 0.0±0.0 vs 0.6±0.9), indicating that embryo quality in all groups was improved. Conclusion: Although it was more beneficial for younger women, AOM administration was very effective to improve oocyte/embryo quality in all age groups. Our present results strongly suggest that AOM can ameliorate age-dependent decrease of embryo quality.